Transcriptome sequencing reveals phagocytosis as the main immune response in the pathogen-challenged sea urchin Strongylocentrotus intermedius.

The clarification of host immune responses to causative bacteria of spotting disease in the sea urchin Strongylocentrotus intermedius is vital to preventing and controlling this disease, especially to selective breeding for disease resistance. For this purpose, sea urchins were challenged with the causative bacterium Vibrio sp. to obtain spotting diseased and undiseased samples. We conducted next-generation sequencing to assess the key genes/pathways in control (CG), diseased (DG), and undiseased (UG) groups. A total of 454.1 million clean reads were obtained and assembled into 23,899 UniGenes with an N50 of 1359 bp, with 86.11% of them matching the genome sequence of the sea urchin S. purpuratus. A total of 8415 UniGenes were mapped to the non-redundant database. Salmon expression analysis revealed 725 significantly differentially expressed genes (DEGs) among CG, DG, and UG. These DEGs were enriched into 72 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including a core set of immune correlated pathways notably in the phagosome, vitamin digestion and absorption, Wnt signaling, and Notch signaling pathways. DG was evidently upregulated in these immune pathways and could enhance phagocytosis directly or indirectly. Thus, phagocytosis was the main coelomic cellular immune response in S. intermedius challenged by spotting disease causative bacterium. The expression patterns of 10 DEGs were confirmed via RT-qPCR, and the expression levels were consistent with the results of RNA-seq. Furthermore, 9899 SSRs were identified, and 123,692, 151,827, and 114,368 candidate SNPs were identified from CG, DG, and UG, respectively. These results provide basic information for our understanding of sea urchin antibacterial immunity.

Vibrio echinoideorum sp. nov., isolated from an epidermal lesion on the test of a green sea urchin (Strongylocentrotus droebachiensis).

A Gram-stain-negative, facultatively anaerobic Vibrio strain, designated NFH.MB010T, was isolated from an epidermal lesion on the test (hard shell skeleton) of a green sea urchin (Strongylocentrotus droebachiensis) collected from northern Norway. Cells of strain NFH.MB010T were rod shaped and motile by means of a single, long polar flagellum. Growth was observed at 1-5% NaCl (w/v) and at 4 °C, but not above 28 °C. Phylogenetic analyses based on eight-gene multilocus sequence analysis (16S rRNA, atpA, gyrB, mreB, pyrH, recA, rpoA and rpoD) suggested novelty at the species level. In silico DNA-DNA hybridization and orthologous average nucleotide identity estimates showed percentage genomic resemblances to its closest relative, Vibrio splendidus, that were well below the established same species threshold values. Phenotypically, utilization of glycogen and gentiobiose, inability of acetoin production, and undetectable valine arylamidase and trypsin activity discriminated strain NFH.MB010T from the closely related reference strains. Protein spectra generated by maldi-tof mass spectrometry further consolidated the species level uniqueness of strain NFH.MB010T. Based on the described polyphasic approach, strain NFH.MB010T therefore appears as a novel species within the Splendidus clade of the genus Vibrio, and the name Vibrio echinoideorum sp. nov. is proposed, with NFH.MB010T (=DSM 107264T=LMG 30656T) as the type strain.

Transcriptome profiling reveals key roles of phagosome and NOD-like receptor pathway in spotting diseased Strongylocentrotus intermedius.

Spotting disease is a common disease in the process of aquaculture and restocking of the sea urchin Strongylocentrotus intermedius and leads to mass mortality. To characterize the molecular processes and candidate genes related to spotting disease in S. intermedius, we conducted next-generation sequencing to assess the key genes/pathways in spotting diseased sea urchin (DUG) compared to healthy ones (HUG). A total of 321.1 million clean reads were obtained and assembled into 93,877 Unigenes with an N50 of 1185 bp, in which 86.48% of them matched to the genome sequence of the sea urchin S. purpuratus and 27,456 Unigenes mapped to Nr database. Salmon expression analysis revealed 1557 significantly differently expressed genes (DEGs) between DUG and HUG. These DEGs were enriched into 151 KEGG pathways including a core set of immune correlated pathways notably in phagosome and NOD-like receptor signaling. DUG displayed an obvious downregulation in these immune pathways. The expression patterns of six DEGs were confirmed by RT-qPCR, and the expressions were consistent with the results of RNA-seq. Furthermore, 15,990 SSRs were identified and a total of 235,249 and 295,567 candidate SNPs were identified from DUG and HUG, respectively. All these results provided basic information for our understanding of spotting disease outbreak in sea urchin.

Colwellia echini sp. nov., an agar- and carrageenan-solubilizing bacterium isolated from sea urchin.

A novel bacterial strain, A3T, was isolated from the intestines of the sea urchin Strongylocentrotus droebachiensis collected in Øresund, Denmark. The strain was Gram-reaction-negative, rod-shaped and facultatively anaerobic, and displayed growth at 5-25 °C (optimum 20 °C), pH 7-9 (optimum at pH 7) and 1-6 % (w/v) NaCl (optimum 3 %). Furthermore, strain A3T grew on agar, agarose, κ-carrageenan, alginate and laminarin as sole carbon source. Complete liquefaction of agar and κ-carrageenan was observed on solid plate media as a result of enzymatic activities. Major fatty acids were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0. The respiratory quinones were determined to be ubiquinones Q-8 (92 %) and Q-7 (8 %), and polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The DNA G+C content was 36.9 mol%. Phylogenetical analyses based on the 16S rRNA gene showed that the bacterium was affiliated with the genus Colwellia within the Alteromonadaceae of the Gammaproteobacteria. The level of 16S rRNA gene sequence similarity between strain A3T and its closest relatives in the genus Colwellia (C. psychrerythraea ATCC 27364T and C. asteriadis KMD 002T) was 97.5 %. The average nucleotide identity between strain A3T and other members of Colwellia was 78.6-80.5 %, and DNA-DNA hybridization prediction revealed values of less than 23 % relatedness between strain A3T and other Colwellia species. The phenotypic, phylogenetic and genomic analyses support the hypothesis that strain A3T represents a novel species of the genus Colwellia, for which the name Colwellia echini sp. nov. is proposed. The type strain is A3T (=LMG 30125T=NCIMB 15095T).

Echinicola strongylocentroti sp. nov., isolated from a sea urchin Strongylocentrotus intermedius.

A yellowish-orange-pigmented marine bacterium, designated MEBiC08714T was isolated from a sea urchin Strongylocentrotus intermedius collected at the west edge of the East Sea of Korea. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that MEBiC08714T was affiliated with the genus Echinicola and that the strain was most closely related to Echinicola vietnamensis KCTC 12713T (96.9 %), followed by 'Echinicola shivajiensis' JCM 17847 (96.3 %), Echinicola jeungdonensis KCTC 23122T (96.1 %), and Echinicola pacifica KCTC 12368T (95.0 %). Cells were strictly aerobic, Gram-stain-negative, rod shaped, flexirubin-type pigments-negative and motile by gliding. Growth was observed at 20-35 °C (optimum 25 °C), at pH 6-11 (optimum pH 7.0), and with 0-13 % NaCl (optimum 2 %). This strain was able to hydrolyze agar and starch. The polar lipids of MEBiC08714T contained phosphatidylethanolamine, an unidentified phospholipid, and four unidentified lipids. The major fatty acids were iso-C15 : 0 (27.5 %), iso-C17 : 0 3-OH (11.5 %), anteiso-C15 : 0 (5.2 %), summed feature 3 (C16:1ω7c and/or C16:1ω6c, 20.3 %) and summed feature 9 (iso-C17 : 1ω9c and/or10-methyl C16 : 06, 6.3 %). The DNA G+C content was 44.2 mol% and the major respiratory quinone was MK-7. On the basis of these polyphasic taxonomic data, MEBiC08714T represents a novel species of the genus Echinicola, for which the name Echinicola strongylocentroti sp. nov. is proposed. The type strain is MEBiC08714T (=KCTC 52052T=JCM 31307T).

Lutibacter holmesii sp. nov., a marine bacterium of the family Flavobacteriaceae isolated from the sea urchin Strongylocentrotus intermedius, and emended description of the genus Lutibacter.

Seven Gram-staining-negative, strictly aerobic, pale-yellow-pigmented, rod-shaped and non-motile strains were isolated from the sea urchin Strongylocentrotus intermedius collected from Troitsa Bay, Sea of Japan. Phylogenetic analyses based on 16S rRNA gene sequences showed that these isolates were affiliated with the family Flavobacteriaceae. The novel isolates showed 99.9-100 % 16S rRNA gene sequence similarity to each other and were closely related to the type strains of the recognized members of the genus Lutibacter with sequence similarities of 95.8-98.4 %. The G+C content of the genomic DNA was 35-36 mol%. DNA-DNA relatedness among the sea urchin isolates was 95-99 % and between strain KMM 6277T and its most closely related type strains, Lutibacter agarilyticus KCTC 23842T and Lutibacter litoralis JCM 13034T, was 38 and 27 %, respectively. The prevalent fatty acids were iso-C15 : 0, anteiso-C15 : 0, summed feature 3 (comprising iso-C15 : 0 2-OH and/or C16 : 1 ω7c fatty acids), iso-C15 : 1 and C15 : 0. The polar lipid profile was composed of the phosphatidylethanolamine, one unknown aminolipid and one unknown lipid. The main respiratory isoprenoid quinone was MK-6.The results of phylogenetic, phenotypic and genotypic analyses indicated that the novel strains represent a novel species within the genus Lutibacter, for which the name Lutibacter holmesii sp. nov. is proposed. The type strain is KMM 6277T ( = CCUG 62221T = LMG 26737T).

Arenicella chitinivorans sp. nov., a gammaproteobacterium isolated from the sea urchin Strongylocentrotus intermedius.

A strictly aerobic, Gram-stain-negative, rod-shaped, non-motile and yellow-pigmented bacterial strain, designated KMM 6208(T), was isolated from a sea urchin. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that this novel isolate was affiliated to the class Gammaproteobacteria and formed a robust cluster with Arenicella xantha KMM 3895(T) with 98.2 % 16S rRNA gene sequence similarity. Strain KMM 6208(T) grew in the presence of 0.5-5 % NaCl and at a temperature range of 4-38 °C. The isolate was oxidase-positive and hydrolysed aesculin, casein, chitin, gelatin, starch and Tweens 40 and 80. The prevalent fatty acids of strain KMM 6208(T) were C16 : 1ω7c, iso-C16 : 0, iso-C18 : 0, C18 : 1ω7c and C16 : 0. The polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and an unidentified aminophospholipid, and the major isoprenoid quinone was Q-8. The DNA G+C content of strain KMM 6208(T) was 46.3 mol%. The DNA-DNA relatedness value of strain KMM 6208(T) with Arenicella xantha KMM 3895(T) was 5 %. Molecular data in a combination with phenotypic findings strongly suggest inclusion of this novel strain in the genus Arenicella as a representative of a novel species for which the name Arenicella chitinivorans sp. nov. is proposed. The type strain is KMM 6208(T) ( = KCTC 12711(T) = LMG 26983(T)).

Altererythrobacter troitsensis sp. nov., isolated from the sea urchin Strongylocentrotus intermedius.

An aerobic, halotolerant, Gram-negative bacterium was isolated from the sea urchin Strongylocentrotus intermedius and subjected to taxonomic characterization. The strain, designated KMM 6042(T), was rod-shaped, motile and yellow-pigmented. Phylogenetic analysis indicated that the strain was most closely related to the type strain of Altererythrobacter dongtanensis, and the level of 16S rRNA gene sequence similarity between the two was 99.0 %. However, the DNA-DNA relatedness between the two strains was 34.4 ± 7.6 %. Physiological and chemotaxonomic properties clearly distinguished the novel strain from other species of the genus Altererythrobacter. It is thus evident from the phylogenetic and phenotypic analyses that strain KMM 6042(T) merits recognition as a novel species of the genus Altererythrobacter, for which the name Altererythrobacter troitsensis sp. nov. (type strain, KMM 6042(T) = KCTC 12303(T) = JCM 17037(T)) is proposed.

Bacillus berkeleyi sp. nov., isolated from the sea urchin Strongylocentrotus intermedius.

A bacterial strain, designated KMM 6244(T), was isolated from the sea urchin Strongylocentrotus intermedius and subjected to a polyphasic taxonomic investigation. The bacterium was found to be heterotrophic, aerobic, non-motile and spore-forming. Comparative phylogenetic analysis based on 16S rRNA gene sequencing placed the marine isolate in the genus Bacillus. The nearest neighbor of strain KMM 6244(T) was Bacillus decolorationis LMG 19507(T) with a 16S rRNA gene sequence similarity of 98.0%. Sequence similarities with the other recognized Bacillus species were less than 96.0%. The results of the DNA-DNA hybridization experiments revealed a low relatedness (37%) of the novel isolate with the type strain of B. decolorationis LMG 19507(T). Strain KMM 6244(T) grew at 4-45°C and with 0-12% NaCl. It produced catalase and oxidase and hydrolyzed aesculin, casein, gelatin and DNA. The predominant fatty acids were anteiso-C(15:0), iso-C(15:0), anteiso-C(17:0), C(15:0), iso-C(16:0) and iso-C(14:0). The DNA G + C content was 39.4 mol%. A combination of phylogenetic, genotypic and phenotypic data clearly indicated that strain KMM 6244(T) represents a novel species in the genus Bacillus, for which the name Bacillus berkeleyi sp. nov. is proposed. The type strain is KMM 6244(T) (KCTC 12718(T) = LMG 26357(T)).

Quality changes in sea urchin (Strongylocentrotus nudus) during storage in artificial seawater saturated with oxygen, nitrogen and air.

BACKGROUND: Sea urchin gonads are highly valued seafood that degenerates rapidly during the storage period. To study the influence of dissolved oxygen concentration on quality changes of sea urchin (Strongylocentrotus nudus) gonads, they were stored in artificial seawater saturated with oxygen, nitrogen or air at 5 ± 1 °C for 12 days. RESULTS: The sensory acceptability limit was 11-12, 6-7 and 7-8 days for gonads with oxygen, nitrogen or air packaging, respectively. Total volatile basic nitrogen (TVB-N) values reached 22.60 ± 1.32, 32.37 ± 1.37 and 24.91 ± 1.54 mg 100 g(-1) for gonads with oxygen, nitrogen or air packaging at the points of near to, exceeding and reaching the limit of sensory acceptability, indicating that TVB-N values of about 25 mg 100 g(-1) should be regarded as the limit of acceptability for sea urchin gonads. Relative ATP content values were 56.55%, 17.36% and 18.75% for gonads with oxygen, nitrogen or air packaging, respectively, on day 2. K-values were 19.37%, 25.05% and 29.02% for gonads with oxygen, nitrogen or air packaging, respectively, on day 2. Both pH and aerobic plate count values showed no significant difference (P > 0.05) for gonads with the three treatments. CONCLUSION: Gonads with oxygen packaging had lower sensory demerit point (P < 0.05) and TVB-N values (P < 0.05), and higher relative ATP content (P < 0.01) and K-values (P < 0.05), than that with nitrogen or air packaging, with an extended shelf life of 4-5 days during storage in artificial seawater at 5 ± 1 °C.

Gramella marina sp. nov., isolated from the sea urchin Strongylocentrotus intermedius.

The taxonomic position of a novel Gram-stain-negative, aerobic, heterotrophic, gliding, yellow-orange-pigmented bacterium, isolated from the sea urchin Strongylocentrotus intermedius and designated strain KMM 6048(T), was established. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that the isolate was a member of the family Flavobacteriaceae affiliated with recognized species of the genus Gramella, forming a distinct lineage within the genus. 16S rRNA gene sequence similarities between strain KMM 6048(T) and the type strains of species of the genus Gramella were 97.4-98.4 %. In line with representative members of the genus Gramella, strain KMM 6048(T) was oxidase- and catalase-positive, hydrolysed gelatin and starch, utilized carbohydrates and possessed a DNA G+C content of 40.0mol%. However, differentiating phenotypic traits and phylogenetic distinctiveness clearly indicated that the strain represented a novel species within the genus Gramella, for which the name Gramella marina sp. nov. is proposed; the type strain is KMM 6048(T) (=KCTC 12366(T)=LMG 25418(T)).

Winogradskyella echinorum sp. nov., a marine bacterium of the family Flavobacteriaceae isolated from the sea urchin Strongylocentrotus intermedius.

The taxonomic position of a novel marine, yellow-pigmented bacterium, designated strain KMM 6211(T), was examined by using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain KMM 6211(T) is a member of the family Flavobacteriaceae, phylum Bacteroidetes. The closest relative of strain KMM 6211(T) was Winogradskyella eximia KMM 3944(T), the sequence similarity being 97.1 %. The DNA G+C content of KMM 6211(T) was 33.6 mol%. The strain was motile by gliding and grew with 1-6 % NaCl and at 4-37 degrees C. Aesculin, casein and gelatin were hydrolysed, but agar, starch, DNA and chitin were not degraded. On the basis of phylogenetic data and phenotypic differences between the isolate and recognized Winogradskyella species, strain KMM 6211(T) represents a novel species of the genus Winogradskyella, for which the name Winogradskyella echinorum sp. nov. is proposed. The type strain is KMM 6211(T) (=KCTC 22026(T)=LMG 24757(T)).

Arenibacter echinorum sp. nov., isolated from the sea urchin Strongylocentrotus intermedius.

Two marine, heterotrophic, aerobic, pigmented and gliding bacteria, isolated from the sea urchin Strongylocentrotus intermedius were subjected to a polyphasic taxonomy study. The 16S rRNA gene sequence analysis revealed that strains KMM 6032T and KMM 6047 formed a distinct branch within the genus Arenibacter, a member of the family Flavobacteriaceae. The level of sequence similarity between the novel isolates and members of the genus Arenibacter was 94.5-98.9%. The DNA G+C content was 39-40 mol%. The dominant fatty acids were iso-C15:1, iso-C15:0, C15:0, C15:1omega6c, iso-C15:0 3-OH, iso-C17:1omega9c, iso-C17:0 3-OH and summed feature 3 (comprising iso-C15:0 2-OH and/or C16:1omega7c). The results of DNA-DNA hybridization experiments supported by phenotypic data indicated that the isolates represent a novel species within the genus Arenibacter, for which the name Arenibacter echinorum sp. nov. is proposed. The type strain is KMM 6032T (=KCTC 22013T=LMG 22574T).

[Distribution of intracellular fucoidan hydrolases among marine bacteria of the family Flavobacteriaceae].

A search for fucoidan-degrading enzymes and other O-glycosylhydrolases has been performed among 51 strains of marine bacteria of the family Flavobacteriaceae isolated from red, green, and brown algae, as well as from the sea urchin Strongylocentrotus intermedius and the holothurian Apostichopus japonicus. Over 40% of the studied strains synthesized fucoidanases. The marine bacteria Mesonia algae KMM 3909(T) (an isolate from green alga Acrosiphonia sonderi), as well as Maribacter sp. KMM 6211 and Gramella sp. KMM 6054 (associants of the sea urchin S. intermedius), were the best producers of fucoidanases. Xylose effectively induced the biosynthesis of fucoidanases in these strains. None of the 15 strains of marine bacteria belonging to the genus Arenibacter produced polysaccharide hydrolases.

Mariniflexile gromovii gen. nov., sp. nov., a gliding bacterium isolated from the sea urchin Strongylocentrotus intermedius.

A marine bacterium, designated strain KMM 6038(T), was subjected to taxonomic analysis via a polyphasic approach. Cells of the strain were heterotrophic, orange-pigmented, Gram-negative and motile by means of gliding. 16S rRNA gene sequence analysis indicated that strain KMM 6038(T) was closely related to the type species of the genera Algibacter and Yeosuana, members of the family Flavobacteriaceae, with sequence similarities of 93.8 and 93.6 % to the respective type strains. However, several chemotaxonomic and phenotypic characteristics, such as the cellular fatty acid profile (iso-C(15 : 0), anteiso-C(15 : 0), iso-C(15 : 1), C(15 : 0), C(15 : 1)omega6c, iso-C(15 : 0) 3-OH and iso-C(17 : 0) 3-OH) and the low G+C content of the DNA (35.7 mol%), indicated that the strain should be separated from these two genera. From the results of phenotypic, genotypic, chemotaxonomic and phylogenetic analyses, the bacterium should be classified as representing a novel genus and species, for which the name Mariniflexile gromovii gen. nov., sp. nov. is proposed. The type strain of Mariniflexile gromovii is KMM 6038(T) (=KCTC 12570(T)=LMG 22578(T)).

Echinicola pacifica gen. nov., sp. nov., a novel flexibacterium isolated from the sea urchin Strongylocentrotus intermedius.

The taxonomic position of three novel marine, heterotrophic, pigmented and agarolytic bacteria with gliding motility, isolated from the sea urchin Strongylocentrotus intermedius, was investigated. 16S rRNA gene sequence analysis revealed that strains KMM 6166, KMM 6172T and KMM 6173 are members of the phylum Bacteroidetes; their nearest neighbours were Belliella baltica and Hongiella marincola (similarities of 94.5 and 93.6 %, respectively). The DNA G+C content of the strains was 44-45 mol%. The predominant fatty acids were C15 : 0 iso, C16:1omega5c, C17:1 iso omega9c, C17:0 iso 3-OH and summed feature 3 (C16:1omega7c and/or C15:0 iso 2-OH). The major respiratory quinone was MK-7. Results of molecular experiments supported by phenotypic and chemotaxonomic data enabled the isolates to be classified as representatives of a novel species in a new genus, for which the name Echinicola pacifica gen. nov., sp. nov. is proposed. Echinicola pacifica is the type species of the genus Echinicola, and its type strain is KMM 6172T (=KCTC 12368T=LMG 23350T).

Roseivirga echinicomitans sp. nov., a novel marine bacterium isolated from the sea urchin Strongylocentrotus intermedius, and emended description of the genus Roseivirga.

A novel strictly aerobic, heterotrophic, pink-pigmented, non-motile, Gram-negative, oxidase-, catalase-, beta-galactosidase- and alkaline phosphatase-positive marine bacterium, designated strain KMM 6058(T), was isolated from the sea urchin Strongylocentrotus intermedius and studied using a polyphasic taxonomic approach. The G+C content of the DNA of the isolate was 41.3 mol%. The predominant fatty acids were i15:1, i15:0, a15:0 and i17:0 3-OH. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain KMM 6058(T) formed a monophyletic clade with Roseivirga ehrenbergii, with 99% similarity. On the basis of phenotypic, chemotaxonomic, genotypic and phylogenetic characteristics, the novel bacterium should be assigned to the genus Roseivirga as Roseivirga echinicomitans sp. nov. The type strain is KMM 6058(T) (=KCTC 12370(T)=LMG 22587(T)).

Reclassification of [Cytophaga] marinoflava Reichenbach 1989 as Leeuwenhoekiella marinoflava gen. nov., comb. nov. and description of Leeuwenhoekiella aequorea sp. nov.

Five heterotrophic, aerobic, halotolerant and pigmented bacterial strains with gliding motility were isolated from Antarctic sea water; one other isolate was collected from the sea urchin Strongylocentrotus intermedius in the Gulf of Peter the Great in the Sea of Japan. 16S rRNA gene sequence analysis indicated that the strains are members of the family Flavobacteriaceae, the nearest neighbour (with 97.1 % sequence similarity) being the misclassified species [Cytophaga] marinoflava. DNA-DNA hybridization experiments and chemotaxonomic and phenotypic analyses demonstrated that the six novel isolates represent a single species distinct from [C.] marinoflava. On the basis of its separate phylogenetic lineage (the nearest neighbours show 92 % sequence similarity), [C.] marinoflava is reclassified as Leeuwenhoekiella marinoflava gen. nov., comb. nov. A second species of this new genus, Leeuwenhoekiella aequorea sp. nov., is proposed for the six novel isolates, with strain LMG 22550(T) (=CCUG 50091(T)) as the type strain.